Microtubules (MTs) are thought to participate in many morphogenetic events by organizing cellular activities. In undifferentiated cells, most MTs are highly dynamic whereas in polarizing or differentiated cells, MTs become stabilized. By understanding how stable MTs participate in the generation of cellular asymmetry, we should gain an understanding of a process fundamental to a wide range of cellular activities since most cells must become polarized to carryout their function. Our overall goal in this project is to determine at the molecular level the mechanism by which MTs become stabilized in directionally migrating fibroblasts and whether the accompanying post-translational detyrosination of tubulin subunits acts as a signal for the association of other organelles (e.g., intermediate filaments [IF] or the endoplasmic reticulum [ER] with stable MTs. We have obtained preliminary evidence that stable MTs are generated by a novel capping mechanism and we will use biochemical and light microscopic assays to identify the putative capping factor. We will also take an electron microscopic approach to determine if distinct structural elements are present on the ends of st able MTs in cells. If we are able to isolate a capping protein, we will make specific antibodies to characterize the subcellular localization and expression of the capping protein further. Our second major goal is to investigate our earlier finding that detyrosinated (Glu) tubulin is a signal for the interaction of IFs with MTs. This signalling hypothesis will be tested by attempting to disrupt the MT-IF interaction with additional antibodies and with soluble Glu epitopes. Whether other IF types and/or other organelles, like the ER, are also signalled by Glu tubulin will also be tested. The factor mediating the interaction between MTs and IFs will be identified by a series of experiments using the putative Glu signal and other reagents as affinity reagents to purify the factor. The possibility that Glu signalling may be important for higher cell functions, such as cell locomotion or neurite outgrowth, will also be tested. Finally, we will initiate a study to determine the relationship of dynamic to stable MTs by examining one hypothesized role for dynamic MTs are formed and then used in cells to create a polarized cytoskeleton. Understanding the role of MTs in locomotion will provide insights into how cells are directed to sites of action during normal tissue responses such as wound healing and may be of direct relevance for pathological processes, such as the metastasis of cancer, where cell motility, becomes unregulated.